The long-range objectives of the proposed study are to determine the role of regulatory T cells in protection against graft-versus-host disease (GVHD) in total lymphoid irradiation/anti-thymocyte serum host conditioning (TLI/ATS), the murine model of a successful human protocol for non-myeloablative bone marrow transplantation (BMT), and to apply this strategy for treatment of thalassemia. This work proposes to further elucidate factors involved in the generation of donor regulatory CD4+CD25+ T cells (CD4+CD25high Tregs) after TLI/ATS, and to determine whether TLI/ATS can be considered to treat thalassemias. This proposal stems from the finding that TLI/ATS-treated hosts given major histocompatibility (MHC)- mismatched BMT are protected from lethal GVHD, whereas TLI/ATS treated NK T cell-deficient and IL-4- deficient hosts develop lethal GVHD, and that BMT from CD4-deficient, IL-4 deficient, and IL-10-deficient donors to wild-type TLI/ATS treated hosts also results in lethal GVHD. In investigating this, we determined that donor CD4+CD25high Tregs are significantly increased after BMT in wild-type TLI/ATS-treated hosts, lost after BMT in NK T cell-deficient hosts, and partially regained upon adoptive transfer of NK T cells to NK T cell-deficient hosts. The hypotheses of this proposal are that: 1) Host regulatory NK T cells mediate GVHD protection via their secretion of IL-4, 2) Host NK T cells allow the maintenance and/or expansion of donor CD4+CD25high Tregs, and 3) TLI/ATS host treatment and allogeneic BMT can allow correction of the thalassemic phenotype without GVHD, in a mouse model of human (3-thalassemia. Using adoptive transfers of purified NK T cells and MHC-mismatched mouse transplants in wild-type and knockout mice, our specific aims are to: 1) Determine the role of host NK T cell IL-4 secretion in inhibition of donor T cell expansion following BMT, 2) Determine the specific donor CD4+ T cell population(s) responsible for GVHD protection, and whether these are regulatory CD4+ T cells, 3) Determine the role of host NK T cells in the generation of donor regulatory CD4+ T cells, and 4) Determine whether BMT after TLI/ATS in thalassemic mice can allow stable donor engraftment without GVHD, and thereby correct the thalassemic phenotype. The proposed work will provide substantial insights into the regulation of donor T cell responses after bone marrow transplantation, and will help define key interactions between innate (NK T) cells and adaptive (CD4*CD25h'9h) immune regulatory cells. This could provide new strategies for the modulation of immune function in treatment of infections, auto-immunity, or cancer, and in transplantation for blood diseases.